CN115261333B

CN115261333B - NT-proBNP hybridoma cell strain, monoclonal antibody, test strip and detection kit

Info

Publication number: CN115261333B
Application number: CN202211171436.8A
Authority: CN
Inventors: 裴世春; 李兴杰; 刘慧宇; 于莲; 赵旻; 张国栋; 赵浚然; 解宇涵; 王戈
Original assignee: Matrell Jilin Biotechnology Co ltd
Current assignee: Matrell Jilin Biotechnology Co ltd
Priority date: 2022-09-26
Filing date: 2022-09-26
Publication date: 2022-12-30
Anticipated expiration: 2042-09-26
Also published as: CN115261333A

Abstract

The invention provides an NT-proBNP hybridoma cell strain, a monoclonal antibody, a test strip and a detection kit. The invention relates to the technical field of biomarker immunodetection, in particular to NT-proBNP hybridoma cell strains, monoclonal antibodies, test strips and a detection kit. The preservation number of the NT-proBNP monoclonal antibody hybridoma cell strain provided by the invention is as follows: CGMCC No.45158. The invention has the technical effects that: the monoclonal antibody secreted by the cell strain has better detection sensitivity to NT-proBNP, can be used for preparing an immunoassay kit and a test strip of the NT-proBNP, and is used for early and rapid diagnosis of cardiovascular and cerebrovascular diseases.

Description

NT-proBNP hybridoma cell strain, monoclonal antibody, test strip and detection kit

Technical Field

The invention belongs to the technical field of biomarker immunodetection, and particularly relates to an NT-proBNP hybridoma cell strain, a monoclonal antibody, a test strip and a detection kit.

Background

Human amino-terminal B-type natriuretic peptide precursor (NT-proBNP) is a linear polypeptide fragment consisting of 76 amino acids with unknown biological activity generated by cardiomyocytes during synthesis of the polypeptide hormone B-type natriuretic peptide (BNP). The Chinese published Chinese cardiac failure diagnosis and treatment guide 2018 recommends screening and intervention on BNP/NT-proBNP for high risk groups (A group) of heart failure, wherein the cutoff value is that NT-proBNP is more than or equal to 125pmol/L or BNP is more than or equal to 35pg/mL.

NT-proBNP is used as a specific marker for detecting heart failure, and the detection method mainly comprises the instant detection technologies (POCT) such as colloidal gold immunochromatography, fluorescence immunochromatography, up-conversion luminescence immunochromatography, magnetic nanoparticle immunochromatography and the like, the enzyme-linked immunosorbent assay (ELISA) technology and other biological detection technologies based on various sensors.

POCT is an extremely efficient, sensitive and rapid detection method, has simple pretreatment on a sample during detection, few steps, low detection cost and simple and convenient operation, is suitable for rapid detection of individuals on site, and is widely applied to the field of disease detection. The test strip is used on the premise that the monoclonal antibody with high specificity and high sensitivity to NT-proBNP is obtained, however, the monoclonal antibody obtained by the conventional method at present usually cannot meet the requirements of POCT detection.

Disclosure of Invention

In view of the above, the invention provides an NT-proBNP hybridoma cell strain, a monoclonal antibody, a test strip and a detection kit, wherein the NT-proBNP monoclonal antibody hybridoma cell strain can secrete the NT-proBNP monoclonal antibody, and has the advantages of high specificity and sensitivity, good stability, low detection cost, convenience in operation and the like.

In order to achieve the above object, the present invention provides an NT-proBNP monoclonal antibody hybridoma cell line, which has been deposited at the common microorganism center of the china committee for culture collection of microorganisms at 20/5/2022, with the deposit name of monoclonal cell line 2A7, and the deposit number: CGMCC No.45158, having a preservation address of No. 3 Hospital No. 1 of Xilu, north Chen, chaozhou, chaoyang.

The second aspect of the invention provides an NT-proBNP monoclonal antibody, which is produced by the NT-proBNP monoclonal antibody hybridoma cell strain.

The third aspect of the invention provides a test strip containing the NT-proBNP monoclonal antibody.

The fourth aspect of the invention provides a detection kit, which contains the NT-proBNP monoclonal antibody hybridoma cell strain or the NT-proBNP monoclonal antibody.

The fifth aspect of the invention provides a detection kit, which contains the test strip.

The beneficial effects obtained by the invention are as follows: the invention utilizes pET-21a (+), pGEX-4T-2 gene vector and BL21 (DE 3) protein expression strain, adopts IPTG to induce and express NT-proBNP, adds GST tag at N end and HIS tag at C end, uses GST tag recombinant whole antigen as immune antigen, HIS tag gene recombinant whole antigen as detection antigen, uses PEG as fusion agent to fuse immune mouse spleen cell and myeloma cell, utilizes HTS-ELISA method to screen fused hybridoma cell, and then carries out multiple subcloning to finally obtain monoclonal antibody hybridoma cell strain, the monoclonal antibody secreted by the hybridoma cell strain has better detection sensitivity (IC) to NT-proBNP ₅₀ The value is 0.078 ng/mL), can be used for preparing an immunoassay kit of NT-proBNP and a colloidal gold test strip, and can be used for early and rapid diagnosis of cardiovascular and cerebrovascular diseases.

Drawings

FIG. 1 is a plasmid map of the complete antigen NT-proBNP-GST;

FIG. 2 is a diagram showing the result of SDS-PAGE of a purified GST-tag protein sample;

FIG. 3 is a diagram showing the result of SDS-PAGE of a sample of purified HIS-tagged proteins.

FIG. 4 is a standard curve for the inhibition of NT-proBNP by the NT-proBNP monoclonal antibody

Detailed Description

The invention discloses an NT-proBNP hybridoma cell strain and application thereof. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to those skilled in the art are deemed to be included within the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

The invention is further illustrated by the following examples.

The reagent consumables and instrumentation required for the experiments referred to in the following examples are shown in the following table:

examples

NT-proBNP is not easy to stimulate mice to generate immune response due to small molecular weight and weak immunogenicity. Therefore, in the embodiment, NT-proBNP is induced and expressed by IPTG through a genetic engineering technology, an HIS tag is added at the C end, a GST tag is added at the N end, and an immune complete antigen NT-proBNP-GST is obtained through gene recombination.

First, a GST tag is added to the N-terminus of NT-proBNP.

The amino acid sequence of NT-proBNP is well known. Wherein, the carrier: pGEX-4T-2, strain: BL21 (DE 3), quality: 35.5kDa. Culturing and inducing the prepared strain to express, comprising the following steps:

s1, culturing escherichia coli: and (3) amplifying and culturing the Escherichia coli BL21 containing the target protein carrier.

S2, induced expression of escherichia coli: expression is usually induced using the IPTG (galactose) induction system.

S3, extracting the escherichia coli expression protein: and (3) crushing escherichia coli and extracting protein.

S4, purifying escherichia coli: purification was performed using a GST-tag purification column.

The detailed process of the steps S1 to S4 is as follows:

about 50ul of glycerol bacterium-bacterium solution (GST tag + NT-proBNP) was extracted using a micropipette (1-200 ul), the bacterium solution and the tip were directly put into a test tube with LB complete medium (5 ml LB complete medium) at 37 degrees, and the tube was shaken overnight for culture (200 rpm).

After overnight incubation, the incubation was continued for 1 hour with 10 times more medium at 37 ℃.

The OD600 of the bacterial liquid reaches 0.5-0.7, and the OD600 is preferably close to 0.6, and the next induction operation can be carried out.

High concentration IPTG (1M) was added to a final concentration of 1mM, and the culture was continued for 4-5 hours at an induction temperature of 30 ℃. Collecting bacterial liquid, centrifuging at 4 deg.C and 4,000g for 20min or at 4 deg.C and 15,000g for 1min, discarding supernatant, and collecting precipitate.

For fresh or thawed bacterial pellets, the lysate is added at a rate of 100ul to 200ul of non-denaturing lysate per ml of collected bacterial pellets, and the cells are resuspended thoroughly. If necessary, an appropriate amount of a protease inhibitor cocktail can be added to the lysate prior to lysing the bacteria.

Adding lysozyme to final concentration of 1mg/ml, mixing, and standing in ice water bath or ice for 30min. Note: lysozyme can be prepared into a mother solution of 100mg/ml by using a lysis solution, and is added before use. The lysozyme is prepared into mother liquor, and can be properly subpackaged and stored at-20 ℃.

Bacteria were lysed ultrasonically on ice. The ultrasonic power is 200-300W, each ultrasonic treatment is 10s, each time interval is 10s, and the ultrasonic treatment is carried out for 6 times. (repeat operation 1 time).

Centrifuging at 10,000g for 20-30min at 4 deg.C, collecting bacterial lysate supernatant, and placing on ice water bath or ice. 20 μ l of the supernatant was used for subsequent detection. Note: the supernatant must be kept clear, i.e. free of any insoluble material, before further purification can be carried out. The purity of the protein obtained by subsequent purification is seriously affected if insoluble impurities are mixed in the supernatant.

1ml of 50% Zhongsonghui GST-tag Purification Resin which is uniformly mixed is centrifuged at 4 ℃ (1000 g multiplied by 10 s) to discard the storage solution, 0.5ml of non-denatured lysate is added to the gel and uniformly mixed to balance the gel, centrifuged at 4 ℃ (1000 g multiplied by 10 s) to discard the solution, and the balance is repeated for 1-2 times to discard the solution. About 4ml of bacterial lysate supernatant was added thereto and slowly shaken on a side-shaking table or a horizontal shaking table at 4 ℃ for 60min-3h.

The mixture of the lysate and Corson-tag Purification Resin was loaded into an affinity chromatography column empty tube provided in the kit.

The bottom of the column was left uncovered, the column was drained by gravity, and approximately 20. Mu.l of flow-through was collected for subsequent analysis.

The column was washed 5 times, 0.5-1ml of non-denaturing wash solution was added each time, and approximately 20. Mu.l of wash solution was collected through the column each time for subsequent analytical testing. SDS-PAGE gels can be used to detect protein in each wash and eluate during column washing and subsequent elution, thereby allowing for increased or decreased washing and elution times.

Eluting the target protein for 6-10 times, and eluting with 0.5ml of non-denatured eluent each time. The eluates were collected into different centrifuge tubes. And collecting the obtained eluent to obtain the purified GST tag protein sample.

FIG. 1 is a plasmid map of the prepared NT-proBNP-GST.

FIG. 2 is a diagram showing the result of SDS-PAGE of a purified GST-tag protein sample.

Secondly, an HIS label is added at the C end of the NT-proBNP.

Wherein, the carrier: pET21, strain: BL21 (DE 3), quality: 9.3KDa.

Culturing and inducing the prepared strain to express, comprising the following steps:

S2, induced expression of escherichia coli: in general, disruption and protein extraction of the E.coli are induced and expressed using IPTG (galactose) induction system.

S3, purifying escherichia coli: purification was performed using an HIS-tagged purification column.

The detailed process of the steps S1 to S3 is as follows:

about 50ul of glycerobacteria-bacteria liquid (NT-proBNP + HIS label) was extracted using a micropipette (1-200 ul), the bacteria liquid and the tip were directly put into a test tube with LB complete medium (5 ml LB complete medium) at 37 degrees, and the shaking table was used for overnight culture (200 rpm).

High concentration IPTG (1M) was added to a final concentration of 1mM, and the culture was continued for 4-5 hours at an induction temperature of 30 ℃.

Collecting bacterial liquid in a centrifuge tube, centrifuging at 4 deg.C and 4,000g for 20min or at 4 deg.C and 15,000g for 1min, discarding supernatant, and collecting precipitate.

Adding lysozyme to final concentration of 1mg/ml, mixing, and standing in ice water bath or ice for 30min. It should be noted that lysozyme can be prepared into 100mg/ml mother liquor with lysis solution, and added just before use. The lysozyme is prepared into mother liquor, and can be properly subpackaged and stored at-20 ℃.

Bacteria were lysed ultrasonically on ice. The ultrasonic power is 200-300W, each ultrasonic treatment is 10s, each time interval is 10s, and the ultrasonic treatment is carried out for 6 times. (repeat 1 time)

Centrifuging at 10,000g for 20-30min at 4 deg.C, collecting bacterial lysate supernatant, and placing on ice water bath or ice. 20 μ l of the supernatant was used for subsequent detection. It should be noted that the supernatant must be kept clear, i.e., free of any insoluble material, for further purification. The purity of the protein obtained by subsequent purification is seriously affected if insoluble impurities are mixed in the supernatant.

Collecting 1ml of mixed 50% Zhongsonghui HIS-tag Purification Resin, centrifuging at 4 deg.C (1000 g × 10 s) to remove the storage solution, adding 0.5ml of non-denaturing lysis solution into the gel, mixing to balance the gel, centrifuging at 4 deg.C (1000 g × 10 s) to remove the solution, repeating the balancing for 1-2 times, and removing the solution. About 4ml of bacterial lysate supernatant was added thereto and slowly shaken on a side-shaking table or a horizontal shaking table at 4 ℃ for 60min-3h.

The mixture of the lysate and the Corson-tag Purification Resin was loaded into an affinity column empty tube provided in the kit.

The bottom of the column was then uncovered, the column was drained by gravity, and approximately 20 μ l of the flow-through was collected for subsequent analysis.

The column was washed 5 times, 0.5-1ml of non-denaturing wash solution was added each time, and approximately 20. Mu.l of wash solution was collected through the column each time for subsequent analytical testing. SDS-PAGE gels can be used to detect protein in each wash and eluate during column washing and the next step of elution, thereby allowing for increased or decreased washing and elution times.

Eluting the target protein for 6-10 times, and eluting with 0.5ml of non-denatured eluent each time. The eluates were collected into different centrifuge tubes. And collecting the obtained eluent to obtain the purified HIS tag protein sample.

Thirdly, the immune animal is fused with the cell.

Among them, SP2/0 cells were purchased from Guangzhou Seiku Biotechnology Co., ltd, and BABL/C mice were purchased from Beijing Wintonliflam laboratory animal technology Co., ltd (license number: SCXK (Kyoto) 2016-0009, SPF grade, male.

(1) Animal immunization

8 BABL/C mice with the age of 6 weeks are selected for immunization, the immunogen is complete antigen NT-proBNP-GST, the immunization dose is 30 mu g/mouse, subcutaneous injection and intraperitoneal injection are carried out, and the immunization is carried out once every two weeks. The primary immunization adopts Freund complete adjuvant to emulsify the complete antigen, and the Freund incomplete adjuvant is adopted to emulsify the complete antigen for 2-3 times of immunization. After 10 days of 3 times of immunization, blood is collected by cutting off the tail, and the serum titer is detected by an indirect ELISA method.

NT-proBNP-HIS was diluted to a concentration of 0.1ng/ml with a coating solution (carbonate buffer), and then applied to an enzyme-labeled plate at a concentration of 100. Mu.l/Kong Bao per well, and the plate was washed with a washing solution (PBS containing 0.05% Tween-20) 3 times at room temperature overnight. Add blocking solution (1% bovine serum albumin solution) 100u l/hole, 37 degrees C temperature 1h incubation, washing 3 times. After the serum sample to be detected is diluted in multiple proportion, 100 mu l/hole is incubated for 1h at 37 ℃ and washed for 3 times. Enzyme-labeled secondary antibody was labeled with 1: after 5000-fold dilution with PBS, 100. Mu.l/well was added, incubated at 37 ℃ for 1 hour, and washed 3 times. Adding TMB substrate solution 100 μ l/well, standing in dark for 10min, adding stop solution (2M H) ₂ SO ₄ ) OD at 450nm was measured by a microplate reader at 50. Mu.l/well. The highest serum dilution when the OD value is 2-fold greater or equal to the OD value of the negative control is the serum titer. Actual detection shows that the average titer of the mouse serum after 3 rd immunization can exceed 1:10 ⁵ The above.

(2) Cell fusion, screening and cloning

Preparation of SP2/O myeloma cells: the culture time is 36-48h before the fusion, the culture benefit is the amplification culture of myeloma cells in the logarithmic growth phase, before the fusion, the cells are blown down from the wall of a cell culture bottle by an elbow suction pipe, collected in a 50ml centrifuge tube, centrifuged at 1200rpm for 4 minutes, and the supernatant is discarded. Then 20ml of 1640 culture solution is added, the mixture is evenly mixed in a heavy suspension way, the mixture is centrifuged for 4 minutes at 1200rpm, the supernatant is discarded, 10ml of 1640 culture solution is added, and the mixture is evenly mixed in a heavy suspension way. 0.1ml of cell suspension is taken, 0.9ml of 1% trypan blue is added, mixed evenly and dripped on a counting plate to count under a microscope for later use.

Preparation of splenocytes: selection of antiserum titers 1:10 ⁵ The above BABL/C mice were immunized 1 time in the abdominal cavity 3 days before fusion, and the immunogen was NT-proBNP-GST,20 ug/mouse, without adjuvant. Before fusion, the eyeballs of the mice are picked and bled, the neck is dislocated and killed, the mice are soaked in 75% alcohol for disinfection, and then the mice are fixed on a mouse board of a biological safety cabinet. Aseptically opening the abdominal cavity, removing spleen, washing twice in a dish containing 10ml of 1640 culture solution, removing peripheral adipose tissue and connective tissue, and placing in a dish containing 10ml of 1640 culture solution1640 medium in a dish. Placing a 200-mesh screen in a plate, adding 1640 culture solution until the screen is immersed, placing the spleen in the culture solution on the screen, and slightly pressing the spleen by using an L-shaped rod to enable spleen cells to completely enter the culture solution. The splenocyte solution is sucked into a 50ml centrifuge tube, centrifuged for 4 minutes at 1400rpm, the supernatant is discarded, 10ml of 1640 culture solution is added, and the mixture is evenly suspended and mixed. Cells were counted as above and ready for use.

Cell fusion: after fusing mouse spleen cells and myeloma cells in 1:1 cell counts, the fused cells were diluted in 1200mL 1640 medium supplemented with 1% HAT and 10% FBS, and then divided into 45 flat-bottomed 96-well cell culture plates at 280 uL/well, and the cells were cultured in 5% CO ₂ And culturing in a sealed incubator at 37 deg.C for 10-12 days. After 10-12 days, 50 μ L/well of the supernatant was transferred to 45 ELISA plates coated with NT-proBNP-HIS and GST, respectively, using a Transtar-96 pipette in a sterile clean bench, and positive hybridoma cells were screened using HTS-ELISA high-throughput assay.

Screening and cloning of positive clones: after 10 days, supernatant is sucked for screening positive holes, the screening adopts adding supernatant into 45 pieces of 96-hole plates of a GST and NT-proBNP-HIS coated plate prepared in advance and then incubating, washing the plate based on classical indirect ELISA, adding 2 antibodies, color developing solution and stop solution, then measuring OD value by using an enzyme labeling instrument, selecting cell holes which show positive in the NT-proBNP-HIS coated plate and show negative in the GST coated plate as a first screening object, respectively sucking each fusion cell colony of the positive holes by using a micromanipulation technology and transferring the fusion cell colony into a new 96-hole culture plate for culturing, performing Confirm-ELISA after the culture medium is discolored, collecting confirmed positive cell strains for culturing, taking cell supernatant of a single cloning hole for antibody detection, continuously performing expanded culture on clones with positive detection results, and freezing and storing the clones for preparing antibodies.

(3) Preparation and purification of monoclonal antibodies

The confirmed positive cell strains are transferred to a 1L rotary bottle for 14 days after amplification culture, culture solution is collected and centrifuged, and then 1:1 and PBS buffer solution, passing through a protein A/G column, eluting with eluent with pH of 3.7, concentrating with ultrafiltration membrane with molecular weight of 3 ten thousand by Amicon stirring type ultrafiltration device, quantifying the concentrated solution with ultramicro ultraviolet spectrophotometer of GE company, packaging, freeze drying and storing.

(4) Determination of the Cross-reactivity Rate between monoclonal antibodies and other proteins

The ELISA plate was coated with NT-proBNP-HIS, 100 ul/well, overnight at 4 ℃, the liquid in the well was decanted, and the washing solution (PBS containing 0.05% Tween 20) was washed 3 times. Add blocking solution (1% bovine serum albumin solution) 100 ul/well, incubate at 37 ℃ for 1h, pour out the well liquid, wash 3 times. NT-proBNP standard substances with different dilution concentrations (100 ng/ml, 10ng/ml, 1ng/ml, 0.1ng/ml, 0.01ng/ml, 0.001ng/ml and 0 ng/ml) and other protein expressions are mixed with an equal volume of monoclonal antibody 1:1 with a moderate concentration (0.1 ug/ml) and added into a coating plate, 100 ul/well is incubated at 37 ℃ for 1h, liquid in the well is poured out and washed for three times. Then, an enzyme-labeled secondary antibody (1-fold dilution, 2000-fold) was added thereto, 100. Mu.l/well was incubated at 37 ℃ for 1 hour, and the mixture was washed 3 times. Adding TMB substrate solution 150 ul/hole, standing in dark for 10min, adding stop solution (2 MH2SO 4) 50 ul/hole, and measuring absorbance (A) of each hole at 450nm by using an ELISA plate. The result shows that the cross reaction rate of the monoclonal antibody secreted by the cell strain of the invention and non-NT-proBNP substances is less than 1 percent.

In conclusion, the invention utilizes pET-21a (+), pGEX-4T-2 gene vector and BL21 (DE 3) protein expression strain, adopts IPTG to induce and express NT-proBNP immune holoantigen with GST tag added at N end and detection antigen with HIS tag added at C end as screening antigen of monoclonal antibody secretion cell strain, and injects immune BALB/C mouse after emulsifying the immune holoantigen with GST tag added at N end by Freund's adjuvant. Selecting mouse spleen cells with high serum titer through three times of immunization and one time of boosting immunization, fusing the mouse spleen cells with high serum titer with mouse myeloma cells through a PEG induced fusion method, and screening out fusion hybridoma cells for specifically recognizing NT-proBNP by using an HTS-ELISA method; and performing subcloning for four times to finally obtain the monoclonal antibody hybridoma cell strain. The monoclonal antibody secreted by the cell strain has better detection sensitivity and IC on NT-proBNP ₅₀ The value is 0.078ng/mL (shown in figure 4), is suitable for the immunoassay of NT-proBNP, and can be further used for preparing an early diagnosis reagent for cardiovascular and cerebrovascular diseasesAnd (5) a box.

In addition, the NT-proBNP monoclonal antibody or the NT-proBNP monoclonal antibody hybridoma cell strain generated by the method disclosed by the invention is used for preparing a test strip or a detection kit containing the NT-proBNP monoclonal antibody hybridoma cell strain or the NT-proBNP monoclonal antibody secreted by the NT-proBNP monoclonal antibody hybridoma cell strain, or the detection kit using the test strip is used for detecting NT-proBNP, and the test strip or the detection kit is not repeated in detail.

The present application has been described in detail with reference to particular embodiments and illustrative examples, but the description is not intended to be construed as limiting the application. Those skilled in the art will appreciate that various equivalent substitutions, modifications or improvements may be made to the embodiments and implementations thereof without departing from the spirit and scope of the present application, and are within the scope of the present application. The protection scope of this application is subject to the appended claims.

Claims

1. An NT-proBNP monoclonal antibody hybridoma cell strain is characterized in that the strain has been preserved in China general microbiological culture Collection center on 5-month and 18-month days 2022 with the preservation name of monoclonal cell strain 2A7 and the preservation number is as follows: CGMCC No.45158, having a preservation address of No. 3 Hospital No. 1 of Xilu, north Chen, chaozhou, chaoyang.

2. An NT-proBNP monoclonal antibody produced by the NT-proBNP monoclonal antibody hybridoma cell line of claim 1.

3. A test strip comprising the NT-proBNP monoclonal antibody of claim 2.

4. A detection kit comprising the NT-proBNP monoclonal antibody hybridoma cell line of claim 1 or the NT-proBNP monoclonal antibody of claim 2.

5. A test kit comprising the test strip of claim 3.